Project Summary: Failure of the immune system to recognize pathogens or abnormal cells can lead to uncontrolled infection or cancer development. With recent successes in manipulating cell function, genetic engineering of T-cells represents a reliable means to enhance T-cell function. However, current methods require T-cells to be engineered ex vivo to recognize specific antigens before they can be infused back into the patient. This approach, while feasible, is labor-intensive, expensive, and requires highly specialized cell production facilities. There is, thus, an urgent need to develop a clinically translatable platform to effectively engineer T-cells directly in vivo. Synthetic delivery methods have, thus far, shown limited potential in targeting T-cells efficiently. To overcome this problem, we have successfully developed an innovative Hybrid EXosomal-POlymeric (HEXPO) nanoparticle platform using naturally-occurring exosomes that display high tropism toward T-cells. We showed that these HEXPO nanoparticles can engineer T-cells at a much higher efficiency than current technologies (>20-fold increase in efficacy). In our research program, we will further employ a systematic approach using different types of naturally occurring exosomes to identify, interrogate, and assess parameters necessary for efficient delivery of genetic materials to T-cells in vivo. We will subsequently use this knowledge to further develop a safe, reliable, and scalable nanoparticle platform capable of re-directing T-cells to specific antigens without the requirement for cell isolation and propagation. We will further examine the therapeutic efficacy of our HEXPO nanotechnology in disease models such as infectious diseases and cancer. The proposed research program is innovative on several levels: First, current research in targeting T-cells have limited efficacy. Our innovative nanoparticle system represents a major advance over existing methods for targeting T- cells. In addition, we have developed methods to scale-up production with the final product suitable for long term storage. This is critical for its clinical translation. Lastly, the idea of directly engineering T-cells in the body to allow them to recognize foreign organisms and/or tumor cells is novel and can completely revolutionize the field of immune cell therapy. The long-term goal of our research program is to provide an innovative approach to harness the power of the immune system to fight a variety of human diseases. In vivo engineering of T-cells using HEXPO nanoparticles offers a major step forward in generating tumor antigen specific T-cells at a low cost with the power of incorporating plasmids encoding any antigen-specific T-cell receptors. The success of our research program will lead to major impact on enhancing immune therapy and improved patient survival for a variety of immune-mediated human diseases. Support through the R35-MIRA mechanism will greatly facilitate this undertaking, which would not otherwise be possible.